Submitted to: Aquatic Fish Health International Symposium
Publication Type: Proceedings
Publication Acceptance Date: 1/1/2003
Publication Date: 2/1/2003
Citation: Overturf, K.E., Lapatra, S.L., Bullock, D.G. 2003. Adenovirus as a method for the deliver and expression of foreign genes in rainbow trout (oncorhynchus mykiss). Aquatic Fish Health International Symposium. Journal of Fish Diseases 2003.p.26:41-101 Interpretive Summary: Currently, transfer of genetic material, whether for targeted gene expression or for DNA vaccines, is limited in fish. For research involving metabolic usage of formulated cereal grain diets, the use of fish expressing elevated levels of specific metabolic enzymes would be advantageous in selection programs. Gene therapy for human diseases have developed a number of methods for delivering foreign DNA into cells. Adenoviral vectors have proven to one of the methods of choice for delivering genetic material into tissue culture cells and live individuals. We decided to test the ability to adenoviral vectors for delivering a marker expressing gene into fish cells and live fish. Two adenoviral vectors were utilized to test gene delivery. One of the adenoviral vectors mainly infects cells that express the adenoviral receptor while the other vector is modified to infect a greater range of cells. In tissue culture both of these vectors demonstrated the ability to infect several tissue lines at 150C, 270C and 370C. Due to differences of viral receptor between cell lines, some cell lines were more readily infectable with the modified virus. Upon immersion of fish in a solution of adenovirus, virus infection was not detected. Injection of the virus directly into the body cavity of live fish demonstrated that muscle displayed the highest level of infectivity with either virus. Direct injection of the virus into muscle showed relatively high rates of viral infection compared to body cavity injection. These findings demonstrate that it is possible to use adenoviral vectors as a means for gene delivery into fish cells or fish for experimental gene expression studies or possibly for vaccine delivery.
Technical Abstract: Fish cell lines were used to assess the feasibility of using adenoviral (Ad) vectors for gene delivery in fish. In view of the fact that most adenoviruses are adapted for infection at 370C, initial experimental infections were performed at 150C, 270C, and 370C to evaluate the infectivity of the virus at temperatures suitable for poikilotherms. Also it was hypothesised that fish cell lines may not express natural receptors for Ad. Thus a tropism modified Ad containing an alternative integrin binding region within the normal Ad binding domain was also used. Both the modified and the native tropism viruses were able to infect fish cell lines. After demonstrating Ad infectivity in vitro, the ability of Ad to infect in vivo was examined. Expression of luciferase (Ad marker) in fry was undetectable when fish were immersed for one hour in PBS containing either Ad vector. Larger fish were also injected intraperitoneally with the Ad vectors. Examination of the individual organs of the ip injected fish detected low levels of luciferase expression in the muscle. To verify that fish muscle tissue could be Ad-infected in vivo, fish were infected by intramuscular injection and the tissue adjacent to the injection site was examined for luciferase expression. In fish injected with either of the Ad vectors expression of luciferase was detected in the muscle tissue. In conclusion it was observed that several teleost cell lines are capable of being infected and it appeared that some cell lines expressed a human serotype adenoviral receptor homologue that aids in Ad infection. Additionally, in vivo studies indicated that muscle tissue of rainbow trout could be infected with Ad vectors, providing an alternative gene delivery strategy in rainbow trout.